New Models to Assay Gene Silencing Therapies

检测基因沉默疗法的新模型

• 批准号: 8234555
• 负责人: DAVID R BORCHELT
• 金额: $ 21.98万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234555
• 关键词: Alzheimer' s Disease; Animal Model; Animals; Antisense DNA; Behavioral; Biological Assay; Bioluminescence; Candidate Disease Gene; Cell Culture Techniques; Cessation of life; Clinical; Code; Codon Nucleotides; Complex; Corpus striatum structure; Critiques; Dementia; Disease; Disease Progression; Disease model; Environment; Equilibrium; Evaluation; Exhibits; Familial Amyotrophic Lateral Sclerosis; Frontotemporal Dementia; Gene Silencing; Genes; Genetic Transcription; Genetic Translation; Glare; Glutamine; Human; Huntington Disease; Imaging Techniques; Impaired cognition; Individual; Investigation; Luciferases; Measurable; Measures; Memory impairment; MicroRNAs; Modeling; Monitor; Motor; Mus; Mutation; Nature; Neurodegenerative Disorders; Neurofibrillary Tangles; Oligonucleotides; Pathologic; Pathology; Phase; Phase I Clinical Trials; Phenotype; Process; Production; Protein Isoforms; Proteins; RNA Interference; RNA Splicing; Reagent; Reporter; Reporting; Research Personnel; Staging; Study Section; Tauopathies; Techniques; Technology; Testing; Therapeutic; Therapy Clinical Trials; Time; Transgenic Mice; Transgenic Organisms; Update; Viral Vector; Work; Writing; base; design; efficacy testing; human Huntingtin protein; in vivo; innovation; knock-down; mRNA Precursor; mRNA Transcript Degradation; meetings; mouse model; mutant; neuron loss; pre-clinical; premature; research clinical testing; tau Proteins; tau aggregation; tau mutation; uptake; vector

DESCRIPTION (provided by applicant): Over the past few years, there has been increasing exploration of the potential for gene silencing or knockdown therapies in the treatment of neurodegenerative disorders. The technology has progressed to a point in which a phase 1 human therapeutic trial for familial amyotrophic lateral sclerosis has been initiated. Current knock-down therapies under investigation include viral vector delivery of shRNAi or microRNA mimics, delivery of naked RNAi and RNAi complexed with various reagents to facilitate uptake, and delivery of modified antisense DNA oligonucleotides. The mechanisms of action for these approaches include modulation of mRNA translation, modulation of pre-mRNA splicing, and degradation of mRNA and pre-mRNA. These various approaches have been tested in pre-clinical animal models to varying extents with varying levels of efficacy. A glaring limitation of these studies that have been conducted thus far is that it has generally been impossible to monitor the efficacy of knock-down in real time. To overcome this limitation, we propose two Aims that are designed to build capability to track the efficacy of knock-down in real time and provide proof of concept studies in mouse models of two neurodegenerative diseases that are potential targets for gene silencing efforts. Taking advantage of the expertise of the investigators involved, we plan to focus on models for Huntington s disease and fronto-temporal dementia. These disorders are prime candidates for gene-silencing therapeutics and previous work in modeling these disorders in mice has produced models that recapitulate aspects of each disorder. In the approach described here, we seek to generate models in which we will produce assayable and observable behavioral phenotypes while simultaneously being able to monitor the efficacy of gene silencing reagents in real-time. In Aim 1, we will generate mice that express mutant forms of human tau fused in-frame to luciferase. In Aim 2, we will similarly generate mice that express mutant Nterminal fragments of huntingtin fused in-frame to luciferase. In both constructs we will employ a technique that facilitates post-translational processing of the poly-protein to liberate the luciferase so it can be assayed independently of pathologic accumulations of mutant tau or huntingtin. We propose to use new in vivo imaging techniques to detect and measure bioluminescence catalyzed by the expressed luciferase. In the tau model we propose to generate, we expect the animals to develop measurable memory deficits with neuropathological abnormalities that include neuronal loss and neurofibrillary tangle pathology. In the Huntington s model, we similarly expect to induce assayable phenotypes, which include motor function deficits, reduction in the transcription of a subset of genes in striatum, hypoactivity, and premature death. Thus, one could ultimately have models with dual readout capability in which reductions in expression could be monitored in real-time by monitoring luciferase activity levels while simultaneously having disease-relevant phenotypes to assay. PUBLIC HEALTH RELEVANCE: The studies proposed in this application are designed to produce and characterize new animal models for pre-clinical testing of gene silencing approaches for intractable human neurodegenerative disease. The innovative models we propose will allow real-time, in vivo, assessments of the efficacy of gene silencing or gene knockdown therapies, allowing investigators to move to Phase I clinical trials with the most optimized and specific therapeutic available. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

描述（由申请人提供）：在过去几年中，对基因沉默或敲低疗法在治疗神经退行性疾病中的潜力进行了越来越多的探索。该技术已经发展到一个点，其中1期人类治疗试验的家族性肌萎缩侧索硬化症已经开始。目前正在研究的敲低疗法包括病毒载体递送shRNAi或微小RNA模拟物、递送裸RNAi和与各种试剂复合以促进摄取的RNAi以及递送修饰的反义DNA寡核苷酸。这些方法的作用机制包括mRNA翻译的调节、前体mRNA剪接的调节以及mRNA和前体mRNA的降解。这些不同的方法已经在临床前动物模型中进行了不同程度的测试，具有不同的功效水平。迄今为止进行的这些研究的一个明显的局限性是，通常不可能在真实的时间内监测敲低的功效。为了克服这一限制，我们提出了两个目的，旨在建立能力，以跟踪在真实的时间敲低的疗效，并提供概念研究的两个神经退行性疾病的小鼠模型，基因沉默的努力是潜在的目标。利用相关研究人员的专业知识，我们计划专注于亨廷顿病和额颞叶痴呆症的模型。这些疾病是基因沉默疗法的主要候选者，并且先前在小鼠中建模这些疾病的工作已经产生了概括每种疾病的各个方面的模型。在这里描述的方法中，我们试图生成模型，在该模型中，我们将产生可测定和可观察的行为表型，同时能够实时监测基因沉默试剂的功效。在目标1中，我们将产生表达与荧光素酶框内融合的人tau的突变形式的小鼠。在目标2中，我们将类似地产生表达与荧光素酶框内融合的亨廷顿蛋白的突变N末端片段的小鼠。在这两种构建体中，我们将采用一种技术，该技术有助于多聚蛋白的翻译后加工以释放荧光素酶，因此可以独立于突变tau或亨廷顿蛋白的病理积累进行测定。我们建议使用新的体内成像技术来检测和测量所表达的荧光素酶催化的生物发光。在我们建议生成的tau模型中，我们预计动物会出现可测量的记忆缺陷和神经病理学异常，包括神经元丢失和神经元缠结 病理在亨廷顿模型中，我们同样期望诱导可测定的表型，包括运动功能缺陷、纹状体中基因子集转录减少、活动减退和过早死亡。因此，最终可以具有具有双重读出能力的模型，其中可以通过监测荧光素酶活性水平来实时监测表达的减少，同时具有待测定的疾病相关表型。 公共卫生相关性：本申请中提出的研究旨在产生和表征新的动物模型，用于难治性人类神经退行性疾病的基因沉默方法的临床前测试。我们提出的创新模型将允许对基因沉默或基因敲减疗法的疗效进行实时体内评估，使研究人员能够使用最优化和最特异的治疗方法进行I期临床试验。 免责声明：请注意，以下评论是由审查员在研究部分会议之前准备的，并且以基本上未经编辑的形式提供。 虽然审查人员有机会根据小组讨论情况更新或修订其书面评价，但不能保证在会议讨论之后更新了个人评论。 因此，这些评论可能并不完全反映小组讨论结束时个人评论员的最终意见或小组的最终多数意见。因此，讨论的简历和摘要是评审员在会议上实际认为关键的最后一句话。